CA1262879A - PROCESS FOR PRODUCING THERMOSTABLE .alpha.-AMYLASES BY CULTURING MICRO-ORGANISMS AT ELEVATED TEMPERATURES - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A process for producing thermostable .alpha.-amylases by culturing micro-organisms at elevated temperatures com-prising the aerobic submerged culturing of a strain selected from the micro-organisms Lactobacillus acidophilus ATCC
31283, Bacillus sp. NCIB 11887 or NCIB 11886, Bacillus circulans ATCC 21822 or any of the mutants thereof, in a suitable culture medium and recovering the thusly produced enzyme from the culture broth.

Description

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The prescnt invention relates -to a process for obtaining an o~-amylase enzyme by the submerged culturing of thermophilic micro-organisms at temperatures of from 50 to 70C, said ~ -amylase enzyme being ob-tained in a state highly resistant -to elevated temperatures even in the presence of low calcium concentrations and under the conditions normally employed in processes for the enzymatic hydrolysis of starch. The preEerred micro-organisms are selected from the group consisting of the species Bacillus circulans, Bacillus licheniEormis, Bacillus sp.

The applications of enæymes in processes ~or hydrolyzing starch have been known for many years, in substitution of the acid catalysed processes which, since they require more severe operating conditions, produce rather negative degradation of the carbonhydrates, ~articularly when the products should be employed for food purposes.

These enzymatic processes generally comprise two steps:
on the one hand, by enzymes classified as d~-amylases, more or less random fragments are produced in the long chain polysaccharides constituting the starch, with a substantial decrease in the viscosity of the starch slurry, known as the li~uefaction step. In the second step, the thusly obtained solution having a low viscosity is treated with enzymes of the amyloglucosidase type which liberate the glucose monosaccharide molecules, which step is consequently known as saccharification.

~' 13'79 This lnven-tion reEers to ~_amy:Lase enzymes, the applications whereof can be very varied, both in processes for hydrolyzlng corn, potato or barley starch, as well as in detergent compositions to promo-te the action thereof on the stains.

The enzyme, for each of these applications, should have certain particular characteristics, wherefore it is rather difficult to propose one enzyme having characteristics which are suitable for all its possible appllcations.

The first commerclally used microbialoC -amylases originated from strains of Bacillus subtilis and the use thereof has been known for many years. However, for the ma~ority of the applications of these enzymes a special emphasis is placed on the stability thereof under elevated temperature conditions or under adverse conditions. In turn~ the effect of the calcium ion as the stabilizing factor of the enzymatic activity is widely described. Hence, the calcium ion concentration is, in many uses, severely limited by the conditions of the process (for example, presence of calcium sequestering-agents in detergent formulations) or the addition thereof produces an economical problem since it should be removed`again, after the liquefaction step, due to its adverse effec-t in the following steps or in the quality of the end product.

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Therefore, it can be sta-ted that at present the requirement of a high thermal stability under low calcium ion conditions constitutes the common denominator of the properties of a commercial ~ -amylase. Some stabilizing factors of the enzymes of B. subtilis to improve the thermal stability thereof under typical conditions of hydrolyzing the starch have been described, for example, in U.S. patent 3,272,717 of J. Fukumoto of Japan, or U.S. patent 3,524,798 in the name of Standard srands Inc. Hydrolyzing process~s operating at temperatures below that of gelatination, in the range of from 60 to 75C, have al~o been claimed, for example, in U.S. patent 3,922,196 and U.S. patent 3,922,200 of C.P.C. However, the most widely accepted processes include a prior gelatination step at 105-110C for about 5 minutes, which requires the presence of oC-amylases which are intrinsically more stable than those of Bacillus subtilis.
Strains producing remarkably more stable enzymes have been descrihed in the latest years. Thus, *or example, British patent No. 1,296,839 of 1972 granted to Novo departed from the Bacillus licheniformis, U.S. patent 3,697,378 of 1972 granted to Glaxo Laboratories departed from sacillus coagulans, and also U.S.
patent 4,284,722 of 1981 granted to CPC International Inc.
departed from ~acillus stearothermophilus, and introducing the culture of a specific micro-organism under thermophilic conditions. However, it should be noted that some enzymes (specifically useful in detergents) are claimed as rather stable at elevated pH, for example in U.S. patent 4,061,541 and U.S.
patent 4,022,666, and others (specially suitable for the hydrolysis of corn starch) are claimed as rather stable under neutral conditions or even under acid conditions (U.S. patent 4,284,722).

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Al-thou~h -there is not an absolute homogenity within each category, there is a remarkabls difference between the conven-tional group of non-thermostale enzyme and the new group of thermostable enzymes. One of the known products of this latter group is commercialized under the registered name of Termamyl of NOVO and origlnates from a Bacillus licheniformis.

The group of conventioncll non-thermostable enæymes is characerized in that its maximum activity is reached between 50 and 65C, while in the thermostable enzymes said maximum activ~ty occurs in the range of from 75 to 85C. Thus, the value of the quotient of the activity at 80C, when compared with the activity at 50C~ will be a good indicator of the thermostability of an enzyme, especially if recorded in the absence of calcium ion.

In the search for new thermostable enzymes, it is reasonable to expect a higher probability of success if research is directed to the group of thermophilic micro-organisms, i.e.
adapted to grow at temperatures of from 50 to 75C, since the protein and enzymatic systems thereof should be better adapted to elevated growing temperatures. In fact, said suspicion is confirmed by reviewing Table I.

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Table I

a) Mesophilic micro-organisms Strain Ratlo activity 80C
(wihtout activity 50C Ca~+) ", ,_ ~
B. Subtilis ~ATCC 6051A) 0.75 B. subtilis (ATCC 21770) 0.28 B. cereus ~ATCC 21769) 0.66 B. amyloliquefaciens ~ATCC 23842) 0.02 B. subtilis (NRRL B 3411) 0.17 B. lichenlformis (NCIB 8059) 0.50 (NCIB 8061) 1.00 " " (ATCC 6598) 1.42 (ATCC 6634) 1.14 " " (ATCC B480) 1.32 ll ll (ATCC 11945) 1.50 b) Thermophilic micro-organisms Strains Ratio activity 80C (without activity 50C Ca~

B. stearothermophilus (ATCC 15951) 1.8 B. stearothermophilus (ATCC 31195) 1.95 B. stearothermophilus ~TCC 31196) 2.10 B. stearothermophilus (ATCC 31197) 2.18 B. stearothermophilus (ATCC 31198) 1.60 B. stearothermophilus ~ATCC 31199) 1.48 Lactobacillus acidophilus ~ATCC 31283~ 1.7 B. circulans (ATCC 21822) 1.7 ~, On the other hand, the exception of the mesophiles comprised of Bacillus licheniformis, studied and claimed, -for example, in British patent 1,296,839 of NOVO, to a range of clutivation temperatures of from 25 to 50C as maxirnum limits, is remarkable, -therefore reising the question as to whether said particular hea-t stability is only and exceptonally stributable to the ~ -amylase produced, or whether the other cellular systems are also specially protected agains-t the high temperatures. In this case, the micro-organism cou]d readily be adapted to be cultured under thermophilic conditions. A first indication in this direction originates from U.S. patent 4,348,480 of 1982 assigned to Miles Laboratories, which descrlbed culturing of a B.
licheniformis in Xylose-enriched media at a temperature of from 50 to 60C for the production of glucose isomerase. It has been discovered that said capacity is not an exception particular of said strain, but other stralns of said species, such as Bacillus licheniformis NCIB 11874 and Bacillus licheniformis ATCC 27811, can be cultivated in simple media to produce dC-amylase, once certain precautions have been taken concerning the adaptation thereof to temperatures in the range of 60C.

~5 .r 8~9 The practical advantages of culturing at high temperatures mainly originate from a saving in the design of the fermentors sincP the cooling sys-tem necessary to remove the fermentation heat a-t 30-35C is almost overcome when operating at 55-60C, and from lesser ease in contamination of the culture and a better maintenance of the sterility copnditions at temperatures above 55C. I t iS not recommendable to increase the temperatures above 60-65C although some micro-organisms present growth at temperatures in the range of 70C since the solubility of the oxygen in the culture medium decrlases with the temperature, and if same is too high large flows o:f air and elevated agltations may be required to avoid low oxygen concentrations in the cellular medium whi.ch would, in general, cause these micro-organisms to direct their metabolisms through an anaerobia via with a slight production of acids, wherefore these working conditions would not be too recommendable for the production of the enæyme.

Besides, the biological processes are accelerated at high temperatures requiring lower cultivation times than loewr temperatures to reach a given enzymatic activity in the broth.
As will be seen from the examples, this reduction in the fermentation time can become very important, permitting large increases in the productivity of the fermentors. Therefore, there is also included within the scope of this invention, the specific process for culturing certain strains of B.
licheniformis at temperatures above 50C for the production of ~-amylase.

Thermostable OC-amylase producing micro-organisms have been selected from different thermophilic bacteria deposited in Micro-organism Collections, for example, NCIB ~National Collection of Industrial Bacteria, Torry Research Station, PØ
Box 31, 135 Abbey Road, Aberdeen AB9 8DE, Scotland), NRRL
(Northern Utilization R search and Development Division, Department of Agriculture, Feroia, Illinois, U.S.A. ) and ATCC

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~American Type Culture Collectlon, 12301 Parklawn Drive, Rockville, Md, 20852, U.S.A.~. As mentioned previously, a higher posibility of finding thermostable ~-amylases was expected in micro-organisms whose growth took place at temperatures above or at 50C than in those optimum growth took place at 25-37C.

To facilitate the search for thermostable ~-amylase producing micro-organisms, a plate activity test was carried out.
The test was based on the halo formed about an dC-amylase producing colony when an iodine solution was poured onto a Petri plate on which a micro-organism has grown from a culture medium containing starch. Specifically, the strain to be tested was reseeded in an AM-8 culture medium ~see Example 1) and it was lncubated for about 48 hours at an optimum growing temperature.
Thereafter, 10 ml of a suspension contlaning 1% ~weight/volume)of starch and 1% ~weight/volume) of agar were poured onto the plate.
The agar was then allowed to solidify and the plates were immediately incubated for 15 minutes at 80C. Thereafter, the plates were cooled and developed using a solution of lugol.
Finally, after 5 minutes' reaction, the sizes of the halos formed about each colony was determined. In general, there was an acceptable correlation between the diameter of the halo and the JC-amylase activity of the colony in question.

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In our search for thermostable dC -amylase producing micro-organsims, we tested 32 thermophilic micro-organlsms corresponding to the following specifies: B. circulans, B.
coagulans, B. licheniformis, B. stearothermophilus, B.
acidophilus, Thermus sp., Microbispora thermodiastatica, Micropolyspora fiaeni, Micropolyspora sp., Thermus thermophilus, Thermoactinomyces vulgarls, Microbispora bispora, ~acillus sp.

This semi-quantitative and rapid test permitted us to select 9 dC-amylase producing micro-organisms whose enzymatic activity was determined according to the following protocol:

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A ~% (weight/volume) solution of starch in a 0.02 M
phosphate buffer, pH 5.7, having 0.006 M Nacl concentration, was heated to about 50C. Likewise the culture broth whose activity had to be determined ~conv0niently dilut~d in the same buffer as the starch~ was h~ated -to the same temperature. Then 0.5 ml of the mentioned enzymatic suspension and 0.5 ml of the starch solution, both previously preheated, were poured into a test tube. The mixture was incubated Eor 5 minutes at 50C under magnetic stirring. The reaction was stopped by sharply droppiny the temperature and adding 1 ml of Bernfeld reagent. Said reagent was prepared as follows: a solution containing 16 gr of Na OH/200 ml of H2O (in any case distilled H2O is used throughout this specification) was poured on 300 ml f ~2 close to boiling point, then 10 g of 3,5-dinitrosalicyclic acid and 300 g of Rochelle salt ~potassium sodium tartrate) were added, the mixture was heated until the reagents were completely dissolved and finally it was leveled -to 1 liter. The samples were then heated in a bath containing boiling water for 5 minutes. After the mentioned period of time, the samples were cooled and 10 ml of H2O were added. Finally, the absorbance of each sample was determined at 540 nm compared with a blank prepared with H2O and the Bernfeld reagent.

A cepsa dC-amylase unit is defined as the mllligrams of reducing sugars, measured as maltose, produced per minute under the previously mentioned testing conditions. Thus:
mg of maltose produced/
Cepsa dC-amylase units/ml of broth = ml..xDxd t where, D: dilution effected in the culture broth; d: dilution effected in the reaction medium; t: reaction time (min.) ,.~

l~Z8~9 Therefore, the absorbance units at 5~0 nm of each sample should be converted into milligrams of reducing sugars measured as maltose. Thus, maltose samples were pr0pared having concentrations of from 0.5 to 5.0 mg/ml and the true content in reducing sugars thereof was determined by the Bernfeld reagent.
Thus, a standard sample was obtained which ~oined absorbance at 540 nm with the reducing sugar content, measured as maltose.
From said standard there was determined, by interpolation, the real content in reducing sugars of each sample, once the A
corresponding to the sugars present in the starch or in the culture broth had been deducted, to the measurement for each case. That is to say, that:
(A540) true = (As40)measured - (A540)starch - (A540)brth It should be taken into accoun-t that the -amylase activity has been determined in all the cases according to this test and, therefore, all the enzymatic units mentioned in the text correspond to cepsa dc-amylase units. The only exception corresponds to the activities measured at 80C (see Table I). In said case, the protocol di~fers only in the temperature used in the reaction of the mentioned enzymatic suspension with the starch, since it is of 80C insteand of 50C. Using this guantitative test to determined the oC-amylase activity and based on the ratio of activities at 80 and 50C (Table I), four micro-or~anisms capable of producing high amounts of thermostableenzymes were selected, in this first approach.

Table 2 illustrates the actiYities of these bacteria, as well as the culturing conditions thereof.

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~LZ~i~8'^~9 Table 2 Culturina Condikions Micro-organism Medium Temp. (C1 Amltation (r.p.m) B. licheniformis AM-76 56 210 B. Licheniformis AM-79 56 210 Bacillus sp. AM-79 56 210 B. Circulans AM-79 56 210 Results obtained under said conditions:
dC -~mylase activity Micro-organism Time (hr) Cepsa ~C -amylase u/ml broth B. Licheniformis 96 172 B.Licheniformis 48 161 Balcillus sp. 24 263 B. Circulans 24 100 - All these cultures were conducted in 250 ml Erlenmeyer flasks containing 100 ml of medium - See Examples 1 and 4 for the composition of the culture media The strains of this invention, and mainly Bacillus sp.
NCIB 11887, have been subjected to the action of different mutagenic agents with the purpose of increasing the ~ -amylase producitivity thereof. Spontaneous mutations during culture ae known, but unfortunately the low frequency thereof prevents the direct selection of super-producing colonies of a particular enzyme. Fortunately, the proportion of mutants can be increased in a colony by using mutagens which induce changes in the bacterial genotype. These mutagenic agents can have a chemical 37~
or a physical nature. Amon~ the chemical agents mainly used by use is N-methyl-N'-nitro-nitrous guanidine (NG). The NG is characerized by its capacity to produce high mutation frequencies even for hlgh survival indexes. The use of NG takes place during the exponential growth of the cul-ture in question, since its maximum effectiven~ss occurs during the replication of the gens.
On the other hand, the ul-tra-violet type radiations have been the maln physical agent used. Its ac-tion mechanism in the inductlon of mutuations in the mentioned micro-organism differs from that attributed to NG. This fact enhances the use in series of both mutagens, thereby obtaining a synergic affect 'n the increase of the ~ -amylase productivity.

In our case, the use of the precisely mentioned mutagens permits a series of characteristics of the strains of the present invention to be improved. Said characteristics are mainly two: production level offf~ -amylase and partial or total unrepression of the synthesis of the enzyme. However, it should be taken into account that the mutants obtained produce amylases whose characteristics coincide with those of the enzyme synthesized by its progenitor, especially, from the point o-E view of the thermostability thereof.

The synthesis of these d;-amylases is clearly influenced by the components of the culture medium. As the ma~ority of hydrolytic enzymes, the production thereof takes place in the final phases of the culture since the synthesis thereof is repressed. In general, an appropriate culture medium should contain a source of assimilable carbon, a source of nitrogen and other necessary nutrients.

It has been observed that there are numerous utilizable sources of carbon: starch, starch hydrolysate, malt extract, maltose, lactose, glycerol, lactic, etc. The concentration used of these carbon sources ranges of from 0.1 to 5% twelght/volume) and preferably of from 1 to 3%. The most suitable sources of ~2 ~

carbon are soluble starch, obtained by the partial acid hydrolysis of po-tato starch. The most appropriate amounts of soluble starch range of from 20 to 40 g/l of culture medium.
Said amounts corresond to cultures in the fermentor, where the pH
is controlled by preventing it from being ad~usted to below 5Ø
When the culture takes place ln erlenmeyer 1asks, the pH is not controlled, wherefore it has a tendency to be acidified and, consequently, the synthesis of dC-amylase is inhiblted. To avoid these problems two measures are aclopted: buffered medla are always used and the concentration of the starch ln the culture medium is reduced to 0.1-1.5% and, preferably to 0.5-1.0%. Under these conditions, the formatlon of acids is minlmized and although the level of synthesized enzyme ls lowered with respect to the fermentors with a higher starch concentration and a controlled pH, it permits the different cultures to be compared readily and economically.

The appropriate nitrogen sources in the culture medlum can be inorganic or organic. The former includes ammonium salts and inorganic nitrates. Sultable organic nitrogen sources includes yeast extract, peptone, hydrolysate of casein, soybean meal, meat extract, corn steep liquor, lactic serum, milk, triptone, cotton seeds, etc. Preferably, the yeast extract or the peptone, in a concentration of from 1.1 to 2% and, preferably, from 0.3 to 0.8%. Besides, the culture media should contain various inorganic salts, such as sodium chloride, magnesium sulphate, calcium chloride, calcium carbonate, etc.
Likewise, cPllular growth and enzymatic productivity can be increased by adding vitamins, aminoacids~ etc., at a trace level.
These sources of carbon and nitrogen, as well as the remaining nutrients, can be used either alone or in combination, as can be seen from the composition of the culture media cited in the Examples.

35The cultivation conditions used for the production of these ~-amylases coincide with those generally used to culture other thermophilic bacteria. The cultures are, preferably, submerged, under aeratLon and agita-tion, at 50-70C, p~ 5-9, for 1 to 5 days.

AS prevlously mentioned, the synthesis o~ dC-amylase takes place when cellular growth has termlnated. From this moment, the enzyme is accumulated in the cultur0 broth. Once the enzyme has been produced, fermentation ls stopped and the broth is cooled to 4C. The cells and other solid residues are then eliminated by filtration or centrifugation of the broth, recovering the d~-amylase in a li~uid phase. This filtrate havingdC-amylase activity ls denominated crude C-amylase in subsequent references ~Example 7). Said crude enzyme can be purified by conventional methods, such as the addition of organic salts or solvents. In the first case, ammonlum sulphate, sodium sulphate, magnesium sulphate, potassium sulphate, sodium citrate, sodium chloride, potassium chloride, etc. can be used. In the event organic solvents are used, the ~C-amylases can be precipltated and recovered by adding methanol, ethanol, ~ 16 '.~ ;Z~
acetone, isop~opa~ol, 1,4-dioxa~e~ etc. Likewise, the ~-am~lase ca~ b~ recovered from the culture broth ~y absorption i~ starch or b~ liquid--liquid extraction i~ mixtures of polyethylene glycol-dextrane. We pre-fer the addition of o_ganic solvents to concentrate a~d purify the crude enzyme. A suitable control of the tem-perature during precipitation prevents the ~am~lase from being denaturalized. The most suitable solvent is ace-tone, the addition of which (6CP/o volume/volume) to the crude enzyme permits precipitation of the c~-amylase. ~he precipitate is recovered by filtration or centrifug~tio~
and it is re-suspended in a 0.05M ~ris-HCl buffer7 ph 7Ø
The average yield of the purifications is above 8CP/o and the purification reached ranges from 1.5 to 2 times with respect to the specific activit~ of the crude enzyme. The final suspensio~ of theCX-amylase in a buf-fer shall be denominated "partially purified ~-amylase"
in subsequent refere~ces (Example 7).
The ~-amylases of this inve~tion were distinguish-ed with respect to the influence of distinct physical fac-tors on the activit~ thereof. The main ph~sical factors which affect the activity and optimum stability~ are ~he pH and temperature9 although other parameters can also affect the reactions. The effects observed in the pro cess, as a result of the variations in the p~ and/or tempera1~re, are due ~oth to the direct i~flue~ce there-o~ on the reaction (ionization7 dissociation, solubili t~, vari.ation in the reaction rate, displacement of the equilibrium, etc.), as well as to the action there=~

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of on the e~z~me itself (Km, VmaX and sta~ilit~).
Figure 1 illu~trates the effect of tne pH. The a~scissa reflects the p~/room temperature units and the ordinate reflects the re:lative activit~ of the.enz~me, accordinK to the standar~l test at 80C, with respect to the activit~ measuL~ed at pH = 5.8~ in the p~esence of about 10-~0 pp~ calcium ion and in a O.lM acetate buffer, for p~'s below 5.7 and a 0.2M phosphate buffer for p~'s above 5.7. Curv- A corresponds to the enzyme of B.
Stearothermophilus 4TCC 3119, curve B to ~. lichenifor-mis NCIB 11874 and curve C to Bacillus sp. ~CIB 11887 and curve D to B. Circulans ATCC 21822. As can be see~
the ~-amylases of this invention, just as that produc~d by B. Stearothermophilus A~CC 31199 descri~ed in U.S.
patent 4,2~97229 seem to be especially suitable for the liquefaction of the starch, since the~ present acti-vity at slightly acid p~ values,, thereby restricting the m~lt~los~ formati~n and facilitate the coupling with the subsequent enzymatic saccharification step~
~ igure 2 illustrates the effect of the'tempera-ture on the activity of the described ~-au~lases. The abscissa reflects the temperatures in degrees centi-grade and the ordin~te reflects the relative activities a~ the corresponding temperature with respect to that obtained in the st2ndard test at 50C. Curves A, Bl C
and D correspond to the same enzymes as figure 1, as well as the buffers used and the calcium ion concentrationO
The ~-am~lases of the present invention, as that con-tained in B. Stearothermophilus ATCC 31199 described i~

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~ '~62~37~1 U.S. patent 47284,722 and Termamyl 120 of Novo, have under these conditions a maximum actiYity at 70-80C-However, from a practical point of view, we should ad~
mit at le~st two types of optimum conditions. ~irstly;
those affecting the maximum activit~, aud secondly those affecting the stability~ ~herefore, a distinction should ~e made ~etween the optimum c~nditions for acti-vity and those for stability which, on the othe~ hand, need not coincide.
The molecular weight of the ~-am~laces of this invention has been determined by electrophoresis. Thus, disk and plate electropkoresis have been carried out on polyacrylamide gel in the presence of sodium dodecyl sulphate (SDS~ The addition o~ SDS permits the pro-teins to be dissociated, wherefore the electrophoretic ~obility thereof5 in the corresponding gels, is a linear function of the logarithm of the molecular weights there-of. ~owever, this method only permits the molecular weight of the denaturalized enz~mes to be obtai~ed.
There~ore, another determination was carried out based on the migration o~ the proteins in acr~lamide gels hav-ing different concentrations. The results of ~oth methods are substantiall~ in agreement. ~encel a mole;
cular weight of 57,000 daltons for ~-amylase of Ba~cillus sp. ~CIB 11887, and 55,000 daltons for that of Baci~llus licheniformis ~CIB 1187~ has been estimated. This value is definitely lower than that of ~-amylase of B. Stearothermophilus ATCC 31199 cited in U.S~ patent 4,2849722.

~ 287 ~ he -tablization of the ~-amylases, in general, ~y Ca~+ has ceen known fo~ many ~ears. However, the negative aspect of the need of said cation as a stabili-zer ~as previousl~ been mentioned. In our case, we have ob~erved that the~-amylases of the present i~vention are sta~le in the prese~ce of low Ca~ levels. Speci~icall~, it has ~ee~ o~served that the hal f life of the ~-amylase, in the absence of a substrate, at high tem-peratures (80 to 100C) is longer than,tha~ correspond r, f~ ~a~ r~
ing to NOVO s Termamyl 120 ~T-120~ a~d similar to that o~ tke ~-amyla~e produced b~ B. Stearothermophilus ATCC
31199 described in U.S. patent 4,284,722, for low Ca+~
~ncentrations. ~able 3 illustrates some of the results o~tained.
T~BLE 3 Type _ Half life (min.~ __ ~ Am~lase Stearothermophilus 8~ 1449 9 Stearothermophilus Bacillus sp. ~CIB
13~4 92 :Bacillus sp. ~CIB

118~7 - Incubation at p~ 5.70 (Tris-maleate buffer) Z87~:~

Thus, it is clear that the ~ amylases of the pre-sent i~vent.ion are very suitable i~ processes for liquefy-i~g ctarch for tke conversion t~e~eof to dextrins. They are speciall~ suita~le for these processes due to the activit~ thereof at low p~ s and high temperatures, as well as due to the high stability thereof even in the prese~ce of ver~ low Ca++ levels.
~ his fact9 as will be seen in the subsequent Ex~
amples, has been conlrirmed b~ h~drolysis carried out on corn starch (Example 8) and soluble potato starch (Ex-ample 7).
Upon hydrolyzing 30-35~0 (weight/volume) suspen-sions of soluble potato starch, at pH 5.7, 80C~ and withou~ exogenous adaition o~ Ca+~, with 4.5 cepsa ~-amylase unitsfromBacillus sp. NCIB 11887 per gram of starch9 a D.E. of 14 16% is o~tained in 3 hours. ~hese valu ~ re J~
similcr to those o~tained with ~ovo s ~ermamyl 120~and the ~amylase of the ~. Stearothermophilus ATCC 31199 des-cribed in U.S. patent 4,284J722 under the same condltions ~ikewise, during the same time, the viscosit~ of the men-tioned starch suspensio~ is reduced ~om about 1.000 cps to less than 20 cps. ~hese, a~d other facts1 confirm that the ~m~lases obtained by the present invention~:are true liquefyin~ kamylases. Furthermore 9 wke~ hydrolyzing 30-35% tweight/volume) corn starch suspenslons with : ~-amylase of Bacillus sp. ~CIB 11887g at pH 5~3 and 185 ppm Ca++, a D.E. of f~om 14-16% is rea~.hed i~ 3 hours~
I~ these cases, h~drol~sis is carried out maintaini~g the reaction mixture ~or 5-10 minutes at 105-110C and , -- cl --1~262~
for 3 hours at ~5C using 4.0 cepsa ~-amylase units/gr of star~hO
~ he following ~xa~ples illustrative of the in-vention a~e not limiting thereof. Obviously cha~ges a~d modifications know~ to the experts in the art can ~e i~troduced t~erein.
Example 1 Four 250 ml erlenmeyer flasks were filled with 100 ml of culture broth having the compositions indicat-ed in the Table. The four flasks were inoculated with a strain of ~acillus licheniformis A~CC 8480. After 7 to 9 dayc' culture at 30C in an i~cubator stirred at 210 r.p.m., tne mentioned activities were obtained in the cul~e broth.
Culture ~-oth AM~ M-3~ biC AM-37 AM-69 Solu~le ctarch (g~'l) ..... 40 40 30 30 Ground Barley ( g/l ) 100 ~arley ext~act (g/l) ...... 100 __ __ Soybean meal (g/l) ........ 30 Pepto~e (g/l) ............. -- - 5 5 Na2S04 ....... ,....... , 1 1 -; --CaC03 (g/l) O.............. 4 4 -_ Ca~12 (g/l) .. ~ 0.4 MnS04 (g~l) ..... Ø...... __ __ 0.5 0.5 MgC12 (g/l) ..... ,........ __ __ 0.5 1-5 ~Cl (g/l)`....... .,....... - -- 0.5 0.5 ~rizma ~ase (g/l) .......... ~ 6 ~ween 80 (~1/1) ,........... 0.1 0.1 -~
P~ = 7.5 .

Cultu-e Broth ~roth activi~
AM ,~3 ....... ~. 34 AM 33 ~is .......................... 14 AM 37 ~ 3 AM 69 .............................. 0 The need to use very concentratsd media contain-ing ground barley or barley extract to achieve sig~
cative activities in the broth when ~he micro-organism is cultu~ed at ~0C, can clearly be seen.
Two 2L fermentors wer e then prepa~ed, one with 1,125 cc of AI1-3~ ~is culture broth and the other with 1,125 cc of AM-6a cultu~e broth containing the previously mentioned composition, to which there were added anti foam agents to prevent the forma~ion of foam; they were sterilized and incoulated with a strain of Bacillus li-cheniformis ATCC 8480. The proportio~ of the inoculum was of 1~.
Growth took place at a temperature of 30C with an aeration of 1~3 v/v/min. aud a stirring speed of 1,750 r.p.m.
~ he ~ests were repeated under similar co~ditio~s with the e~ception of the culturi~g temperature which was raised to 56C and the inoculum used, which was a strain of Bacillus licheniformis ATCC 8480t adapted to grow at high te3peratures. The proportion of inoculum u~ed was also of 10~.
The inoculum for the fermentor co~taining AM-69 culture medium was prepared as follows: ~he strain of Bacillus licbeniformis ATCC 8480 was re-seeded in~an inclined AM-8 a~ar tu~e~ it was cultured at 56C for 48 il7'~

hours . The cul~ure grown in AM- 8 was washed with a solu tion of 9~/0 NaCl and the thusly obtained suspension serv-ed as the inoculum for a 250 ml erlenmeyer flask contain-ing 100 ml of the previously mentioned AM-33. Incuba-tion took place for 14 days at 56C and 210 r.p. m. 14 days thereafter, 10 ml o.f this culture served as the ino-culum fo. a 250 ml erlenmeyer fla~k co~taining 100 ml of the ~M-76 culture medium.. It was incuba-ted at 56C for 72 hours at 210 r.~.m. 'rhis culture ~erved as the inocu-lum for the fermentor.
rrhe com~osition of the AM 76 culture medium was the following:
Solu~le starch (g/l) ..... ....5 Peptone (g/l) ......... l.. ....5 CaC12 (g~l) .............. ....1 Iln S04 ( g/l ) .......... ...Ø5 Mg C12 (g/l) ---~........ 0~. 1.5 E Cl (g/l) ............ ......... 0.5 ~rizma base (g/l) ................ 6 pH = 7.5 ~ he inoculum for the fermentor containing AM 33 cultuL~e medium was prepared following th~ first two steps of the preceding case. After 14 days'growth 10 ml of culture served as the inoculum for a 250 ml erlenme~er flask containing 100 ml of AM-33 culture medium, it ~as incu~ated fox 72 hou~s at 210 r.p.m. at 56C and this cul$ure served as the inoculum for the fermen$or.
AM-8 culture medium

- 2~ -~ ~2~37~

Starch (g,il) ........ '0 r~eptone (~/1`, ~..... 10 Ag~r-a~,ar (g/l) ..... 20 Growth took place at 56C with an aeration of 1,3 ~/v~min. a~,d a stirring speed of 1,750 r.p.m~
~he re~ults o~tained i~ the two tests a~e as follo~s:
Tem erat~e C
~0 5~
CultuLreActivit~ Time of Activity ~ime of ~,ediumu/CEPSAj~,l. ~ ~ . ~ ) ~M 33 bis1~ 164 45 113 It is clearl~ demonstrated that at a high tempera-ture, contrary to what takes place at 2~0C, the poor medium not containing ~arle~ is tke most adequate for the produc-tion of ~,-a~ylase, furthermore obtaining a substa~tial saving in t~e culturing time~ Th~ls9 the adaptatio~ to high temperatures not only inc~eases the speed of growth, ~ut also produces a substantial alteration of the meta~
bolic pattern of the micro-organism. ,,' ,xample 2 ' Three 250 ml erlenme~er flasks were filled with 100 ml of AM 76 culture broth described i~ Example 1.
The three flasks were inoculated with a strain of Bac~
lus licheniformis ATCC 27811 and incubated at te~pera- `
tures of 2~0~ 45 and 56C at 210 r.p.~. ~fter 3 4 days~ 1, culture, the following results were obtained:

~, i , ~Z~
Tem~ferature (C) Activity Time of ~oth (CEPSA u/ml) Cult~Lre (h) ~ _ . .

~ 96 As in the case of the strain of Bacillus liche~i-forDis ATCC f~48fO, the productio~ of,?,amylase increased as the culturing temperat~re of the micro organism in-creased.
Exam?le 3 A 2L ferme~tor was filled with 1,125 cc of the AM-69 culture medium described in ~amplb 1, to which there was added an antifoam agent to prevent the forma-tion of foam, it was sterilized and inoculated with a culture of tke strain of ~f'facillus licheniformis NCIB ifi 11874~f capa~le of growing at high temperatures. The inoculum was prepared followin,g the procedu~ffe described in Example 1. 'Ihe culture was carried out at a tempera-tLre of 60C, aeration of 1,3 v/v/min and a pH of 7.5 1~
with a stirri~g speed of 19750 r.p.m. Under these con- , ditions and 40 hours thereafter~ an activity in the cul-ture broth of 270 cepsa units/ml. was reached.
Example 4 ,1 , A 2L ferme~tor was filled with 1,125 cc~of AM-96 5 culture medium to which an antifoam agent was added to pre~ent the formation of foam, it was sterilized and inoculated with a cultuxe of the strain of Bacillus lic~enifo:rmis NCIB 11874~ capable of gro~ing at high ¦, te~pferatuxes. The inoculum was prepared as described ;, ., , .

21~7'~

in ~xample 1, with the exception that the AM-79 instead of the AM-76 culture ~ediu~ was used.
The ,culture was carried out at a temperature of 60C, aeration of 1,3 v/v/min, and a p~ of 7.5 with a stirring speed of 1,750 r.p~m. Under these conditions and 63 hours thereafter, an activity in the culture broth of 317 cepsa u~it/ml. was reached~
Composition of the culture broths:

__ ___ Soluble starch (g/l) 5 30 Yeast extract (g/l) 5.07 5.07 Ca C12 (g/l) Mn S04 (g/l) 0.5 0.5 Mg C12 (g/l) 1~5 1.5 E Cl (g/1) o.5 o.5 Trizma base (g/l) 6 6 pH = 7~5 ; Four fer~entors containing AM-69 culture ~edinm~described in Example 1) were inoculated with the follow-ing strains: ~acillus stearothermophilus A~CC 31199g Bacillus lichenifor~is NCI~ 118749 Bacillus sp. ~IB
11887 and Bacillus circulans ~TCC 21822. ~he culturing conditions used, in all cases, were the following. 56C
temperature, pH 7.5, aeration 1,3 v/v/min. and a stir-ring speed of 1,750 r-p.m. After 60-80 hours' cultur-ing same was stopped~ the broth was centrifuged to re-move the cellular debris and the heat stability of the ~-am~lases produced was tested.

~26~'7 The process used to determine the heat stabi-lity of these c~-a~ylases will now be described. The culture broth was diluted in a ~ris-maleate buffer9 pH
5.70, so that the final concentration of the enz~me was Of about 4 cepsa units/ml~ of solution. ~t the same time, the concentration of Ca~+ ions was adjusted to 5.25 or 36 ppm, depending on the test. The estima-tion of the residual act:ivity in each ca~e, after i~-cubation for 15 minutes at 80C, permitted the follow-ing values to be obtained:
~L~
Ca+~ (ppm) 5 25 36 Enzyme origin:
B. Stearothermophilus ATCC 31199 4~ ~~ 71 B~ licheniformis NCIB

Bacillus sp ~CIR 1188734 56 77 B. circulans ATCC 218?2 45 54 --Whereas 9 under these conditio~ ov0'9 Termamyl 120 retained 80% of its initial activit~ in the presence of 36 ppm Ca +, but it wa~ c ompletel;~ deactivated, e~en after 10 minutes' incubation, i~ only 5 ppm Ca~ were added.
If the sa~e test is repeated, but i:~cubati~g the ~-a~;ylases at 90~C, the results obtained 15 ;minutes thereafter are the following: ~ ~

-- 2~3 _ t7 ~99~
Ca++ ~ppm) 25 36 Enzy~e origin:
B. Stearothermophilus ATCC ~1199 -- 50 B~ liche~iformis NCIB 11874 -- 54 Bacillus sp. NCIB 118&7 17 46 B. circulans ATCC 21822 19 --Whereas~ under these sa~e conditions, ~ovo's Termamyl 120 retains 17% of it5 initial activity i~ the prese~ce of 35 ppm Ca++~
Example 6 A 2L fermentor was filled with 1,125 cc of the AM-69 culture medium, it was sterilized and inoculated with a culture o~ the strain ~acillus circulans ATCC
21822~ ~he proportion o~ the inoculum was of 10%. The culturing conditions for the inoculum grow~ in AM-76 were of from 20 to 24 hours at 56C and at 210 r.p.m.
The culture in the fermentor was carried out at a tem-perature of 56C, aeration of 1,3 v/v~min. a~d a p~ o~
7.5 with a stirri~g speed of 1,750 r~p.m. Before sterilizing the medium, an antifvam age~t was added to prevent the formation of foam~ ~uder these conditions and 48 hours thereafter, an activit~ in the culture me-dium of ?oO cepsa units/ml. was reached.
Example 7 ~ he culture broths from different ~-amylase produce:rs, obtained as described iu E~ample 3 were used to hydrolyze a soluble 33~ potato starch solutio~ (weight~
volume~ i~ a Tris-maleate buffer, p~ 5.70~ Thus, a 1~6Z87 starch suspension was prepared in the least possible a~ount of the mentioned buffer. Said suspension was poured onto a boiling buffer, boiling and stirring ~ere maintained until the complete solubilizatio~ of the starch. Then 1~5 cepsa ~-a~ylase units/ml. of starch solution were added7 equivalent to 4.55 cepsa ~-amylase units/g o~ starch~ ~ydrolysis of the starch was imme-diately initiated at 80C.
This h~drolysis process is co~ducted with crude or partiall~ purified enzymes. In the first case, the ~-a~ylases contained in the culture broth were directl~
U52~, while i~ the second case the ~-am~lases used ori-ginated from a purification process with organic 801-vents~ ~herefore, the ~amylases present in the broths were precipitated with 60~ (volume/volume) of acetone, at 0C and under stirring. The thusly obtai~ed precipitates were re-suspended in ~ris-ECl buffer9 pH 7Ø
The h~drolytic process was controlled with D.E.
~dextrose equivalent) anal~sis and distribution o~ oligo-mers in the differe~t samples. The D.~. was determined according to the Lane-Eynon method, while the different degrees of pol~merization of the oligomers resultin~ from the hydrol~sis was calculated by high pressure liquid-liquid chromatography. Table~ 5 and 6 summarize the values obtained for the different tgpes of ~-amylases.
As can be observed in Table 5 there are no substant;ial differences, with respect to the evolution of the D~E. (%) in the h~drolysates 9 between the dlf-ferent t~pes of ~-am~lases testedO Likewise, substan-tial differences are not detected as a result Or the use :~LZ6,~8~7 o~ crud~ or partially puxified enzyme.

____ __ ~ stearoterm.

a) Crude 6.7 8~7 807 11.1 14.5 b) Purified 4~3 6.5 9.0 11~3 15.2 B. liche~iformis ~CrB 11874 a) Crude 3~9 7-5 8~7 8.7 15.5 b) Purified 674 8.2 10.5 13.4 15.4 Bacillus sp .

a) Crude 6.4 8.9 10.~ 13.2 15.4 b) purified 7.0 11.1 13.0 15.5 1~.8 ~. circulans a) Grude 3~3 7.6 9.3 llr? 1~o6 b) Purified 4.0 8.1 9.5 12.2 1400 T-120 Novo 6~2 9.0 1104 13.1 15.1 :Bacillus sp.
NCI~ 86 : a) Purified 7.0 11.8 12.9 16~1 1608 0~ the other hand, ~able 6 illustrates that the distribu~ion of oligomers in the hydrol~sates dif~er ac- ¦
cording to the type of c~-am~lase usedO IA general, the o(-amylas~ss produced by Bacillus licheniformis, 8UCll as i~CIB 11874 and Novo T-120 ;yield ~ higher proportiorl OI
oligomers haYi~g a low molecular weight than the re~ain-8'79ing ~nz~mes.
Nevertheless, the slight differences appearing in the distri~utions oL oligomers of the different enzy~es, do not noticeabl~ affect the ~ields of the industrial ap-plication thereof.
TAB~E 6 T,~e of 1 2 DP3D~,, D~5 DP6 DP7 ~P
E;~drol~Tsis (~ ) (%) ~;~) (~) (;;, (Rlin.) T-"20 60 1.~ ~.67.65'9 5.610.8 4.8 59.3 !;01l0 120 2.0 a.s13.18.3 9.l13.9 11~.2 30.6 .stearoterm. 60 1.24.17.2 3.9 3.4 13.~ 4.6 52~S
ATCC-j199 1Z0 2.6 6.99.61~.1 4.6~0.5 0.2 51~5

3.1ich~nif.60 0.6 3.39.9b.9 10.5 9,4 0.6 60.8 ~CIB 1187~ 120 0.8 5.212.26.115.110.3 0.3 50.0 1 6acillus sp. 60 ~razas1.7 6.3 1.3 3.5 12.1 9.9 65.2 ~ l5CIB 11887 lZ0 lra~as1~.6 8.3 1.6 9.1 17.1 8.2 51.1 ¦ 8.circulans 60 7~a2as2.3 6.0 2.8 3.1 10.7 9.5 65.6 !ATCC 218221Z0~razas 3.97.21.5 3.713.8 7.3 52~6 - '1 ~ j;
Various ~ am~lases producing strains were cul-t~red as described i~ Example 5. ~hen the correspo~di~g enzymes were purified and recovered according to the pro-cedure described in Example 7. ~he resulting ~-a~ylases !
were tested i~ the hydrol~sis of corn starch, under con-ditions similar to those used i~dustriallyO ~hus, dif-ferent aqueous starch solutions were prepared, varying the Ca~+ concentration ~by addin~ for example Ca C12), the p~ (ad~usted with ~aO~) and the conce~tration of 126~t37 the starch~ I~ all these cases 4 cepsa ~-amylase units/
g of h~drolyzed starch were usedO The evolution Or the temperature during the p:rocess took place as follows:
i~mediately after adding the en2yme, the ~uspension ~as rapidly heated to 105-110C, this te~perature was then mai~tained for 5 minutes and finally the suspensio~ was cooled to 95C, at which temperature the mi~ture was mai~-tained for 3 hours.
In the event suspensionfi containing 43.5% (weight/
volume) of starch were h~drolyzed in the presence of 185 ppm Ca+~, the final D.E. (%) for each case were the fol-lowing:
~D~o~he ri~
. stearothermophilus CC 31199 13~ 5-i B. licheniformis NCIB
11874 14.6 5.7 Bacillus sp ~C B 11887 16.5 5~7 T-120 ~ovo 18.7 6.2 with 4~o starch suspensio~s and a hydrolysis p~ of about ¦~
5.4, the final D.E. (%) for di~ferent Ca++ conce~tratio~s t were the following: i I

B. stearother~ophilus ATCC
31199 17.3 12.2 B. licheniformis NCIB 11874 18.9 8,7 ~acillus sp. NCIB 118~7 17.7 12.2 B. circulans ATCC 21822 --- 12.8 T-120 ~Jovo 20~ 3 Finally, when the suspe~sion containing 35%
(weight/~Jolu~e) of starch was hydrolyzed, in the pre~ence ~2~79 of 18C pp~ Ca+~, at a pH of 5.3, the followin~ r0sults were obtained:

~. stearothermophilus ~TCG 31199 8.5 B. lichenlfor~is ~CIB 11874 1~.5 Bacillus sp. NCIB 11887 15.5 B. circulans ATCC 21822 120 5 T-120 ~ovo 7.1 A 2L fer~entor was filled with 1,125 cc of the AM-69 culture broth, to which a~ a~tifoam agent was added to pre7ent the for~ation of foam, ~he thusly prepared fermentor was sterilized and inoculated with the AM-76 culture medium described in Exa~ple 1 of the strain Bacil- !
lus sp. ~CIB 11887. The culturing conditions for the i~oculum were of 20 hours at 56C a~d 210 r.p.m. The :~
culture in the fer~e~tor was carried out at a tempera~ur~ ~
of 60C, aeration of 1.3 v/v/min., p~ 7.5, and a ~tirring .
speed of 1,750 rOp.m~ ~nder these conditio~s a~ activi-t~ of 380 cepsa u~its/ml~ were obtai~ed 53 hours after culturing.
~0 ' .
~ he straiu of Bacillu~ sp. NCIB 11887 was treat- .
ed with NG to obtain muta~ts. ~rom the 350 colonies ob-tained, after measuri~g the ~-amylase activity, the ~trai~
3666 (registered in the ~CIB u~der ~o~ 11886~ was select-ed. .
Strain 3666 was cultivated in two 2~ fermentors:
one containing 1,125 cc of the AM-69 culture medium and~
the otker containing 1~125 cc of the AM-96 culture me- I

- ~4 -dium described in EXample 4. ~u antifoa~ agent was ad-ded to the two fermentors to prevent the for~ation of foam. One of the thusl~ prepared fermentor~ was i~oculat-ed with a culture of the strain 3665 grown in AM-76 d~s-cribed i~ E~-a~ple 1 and the other with a culture of strain 3666 grown in AM-79 described i~ E~ample 4~ respectively.
The culturing conditions of the inoculums were: 20 hours at 56C and 210 r.p~.
Cultu~ing in ferme~tors too~ place at a tempera-ture of 60C, aeration of 1.3 v/v/min, and a stirring speed of 1,750 r.p.m. ~nder these co~ditions the follo~-ing results were obtained: !
Culture MediumActivit~ Culturi~g time ~Q

AM-96 40Ci 44 ~ .
Six 2~ ferme~tors were filled with 1,125 cc of the AM-96 culture medium to which an aDtifoam agent was added to preve~t the formatio~ of foam. ~hey were steri-lized and inoculated with a cult~re of strai~ 3666 grown in AM-76 described in Example 4. The culturing condi- ¦
tions for the inoculum were of 20 hour~ at 56C and 210 r.p.m. Culturing in ~e ferme~tor was carried out u~der the following conditions: Aeratio~ 1.3 v/v/min., stir-ring speed 1,750 r.p.m. In three of the ferme~tors the i~luence of the tenperature was studied and the p~ ~as o~ 7.5~ t;he temperatures to bei studied being of 55, 60, 65C. In the three remaining fermentors, the influence ~6~7 c~ the pH was studied, the culturing temperature being of 60C and the p~I to be studied of 6.5, 7.5 and 8.
Under these conditions the followirlg results were ob-tai~ed:
Batch Fed Batch Temp. p~I Culturing ActivityCulturing Acti~ity C Time (h)Cep~;a u/ml. Time (h)Cepsa u/ml.
___ __ __ 7.5 68 290 57 lg7 7.5 68 385 6~ 2~4 7~5 68 52 69 154 6.5 40 156 34 325 7.5 40 21 5 34 423 Two cultures of the micro-organism B. liche~
formis NCI~ 11874~Bacillus sp. NCIB 11887 were prepared in 250 ml erle~meyer flasks containing 100 ml of the AM-79 culture medium (See ~xample 4). The cultures were incubated at 56~C for 72 hcurs in a~ orbital stirrer (210 r.p~mO)~ ~t the end of the culture~ the e~z~mes~
w~re recovered folllowing the procedure described in Ex- ¦
ample 7. The partially purified enzymes, using ace~one, were subsequentl~ purified by precipitation of the cor-respo~ding enzyme-starch complexes, as described by Michael Schram and Abraham Loyter in "Methods in Enzy- .
~ology" Vol. VIII, pages 533-5370 ~he purified enzyme~
were subjected to electrophoresis i~ polyacrylamide gels with sodium dodecyl sulphate to deter3ine the molecular ~eight thereof. For this purpose the tech~ique describ-ed by E. Weber and M. Osborn in J. Biol. Chem~ 244, ~6 .

87~

4406 (1969) W~8 used, and albumina (P.M. 67000), catalase (P.M. 60000) ovalbumina (P.M. 43000) and LDH (P.M.. ~6000 were used. This technique permits a molecular weight of 55,ooo daltons for the ~--amylase of B. liche~iformis NCI:B
11874 and of 57,000 daltons :For :Bacillus sp. NC~ 11887, to be determined.

Claims (8)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for producing thermostable .alpha.-amylases by culturing microorganisms at elevated temperature comprising the aerobic submerged culturing of a strain selected from the microorganisms Lactobacillus acidophillus ATCC 31283, Bacillus sp.
NCIB 11887 or NCIB 11886, Bacillus circulans ATCC 21822 or any of the mutants thereof, in a suitable culture medium at a tempera-ture of 50 to 70°C and at a pH of from 5 to 9 for 1 to 5 days and recovering the thusly produced enzyme from the culture broth.

2. A process according to claim 1, in which the strain is selected from the microorganisms Bacillus sp. NCIB 11887 or NCIB 11886 or any of the mutants thereof

3. A process according to claim 2, comprising the cul-ture of a strain of Bacillus sp. selected from NCIB 11887, NCIB
11886 or any of the mutants thereof, characterized in that the culture medium used contains a source of carbon, a source or organic nitrogen and suitable trace substances.

4. A process according to claim 3, wherein the pH is from 5 to 8.

5. A process according to claim 1, 2 or 3, wherein the culturing temperature is from 55°C to 65°C.

6. A process according to claim 3, wherein the source of carbon is selected from starch or its hydrolysates having a concentration of from 5 to 60 g/l, the source or organic nitrogen being selected from the group consisting of peptone, yeast extract and corn steep liquor.

7. A process according to claim 3, wherein the cultur-ing temperature is from 55°C to 65°C and the pH is from 7 to 7.5.

8. A thermostable .alpha.-amylase obtained by a process according to claim 3, characterized by the following characteristics: a molecular weight, measured by electrophoresis in a polyacrylamide gel, of 57,000 daltons; suspended in a tris-maleate butter, pH 5.7, and at 80°C, it has a half-life with respect to its activity of about 44 minutes in the presence of 36 ppm calcium ion, and of about 92 minutes in the presence of 87 ppm calcium ion; a maximum activity temperature at pH 5.7 and with 10 to 20 ppm calcium of about 80°C.